We determined the hemolytic properties of the LTZ @ GA-AuNPs to evaluate the interaction between the nanoparticles and blood components. Results showed that there is no interaction between LTZ @ GA-AuNPs and blood. In conclusion, the findings indicate that LTZ @ GA-AuNPs has significant potential as a promising drug delivery carrier for treating breast cancer in postmenopausal women.The most prevalent malignancy among postmenopausal women is breast cancer. It is one of the leading causes of cancer-related mortality among women. Letrozole (LTZ) is a clinically approved inhibitor for breast cancer in postmenopausal women. However, due to poor aqueous solubility, non-specific binding, unwanted toxicity, and poor blood circulation hampered its clinical applications. To maximize the pharmacological effects and minimize the side effects, inorganic nanoparticles are a good alternative. Due to excellent biocompatibility and minimum cytotoxicity, gold nanoparticles (AuNPs) offer distinct benefits over other metal nanoparticles. Emerging as attractive components, AuNPs and Gum acacia (GA) have been extensively studied as biologically safe nanomaterials for the treatment of cancers. This study reports the synthesis and characterization of GA stabilized gold nanoparticles (GA-AuNPs) of LTZ for breast cancer treatment. The observed particle size of optimized LTZ @ GA-AuNPs was 81.81 ± 4.24 nm in size, 0.286 ± 0.143 of polydispersity index (PDI) and −14.6 ± −0.73 mV zeta potential. The biologically synthesized LTZ @ GA-AuNPs also demonstrated dose-dependent cytotoxicity against the human breast cancer cell line MCF-7, with an inhibitory concentration (IC Breast cancer, in its advanced stages, is one of the leading causes of cancer-related fatalities in women worldwide, mainly in the United States ,2. Most Letrozole (LTZ) is an orally available third-generation aromatase inhibitor that works as an efficient non-steroidal anticancer drug . The facThe half-life of LTZ is two days, and its plasma concentrations reach a steady state in an average of 2 to 6 weeks after being administered . AlthougMeanwhile, researchers are searching for vehicles that would deliver drugs precisely to the targeted organ or tissue. A delivery mechanism that can permeate both the cellular and outer mitochondrial membranes is being developed by scientists to induce apoptosis more efficiently and effectively. The targeted drug delivery system based on nanoparticles has gained considerable attention in cancer treatment during the last few decades ,20,21. IGold nanoparticles  are among the most widely utilized inorganic nanoparticles in biomedical applications because of their excellent compatibility with the biological system, small size, reduced toxicity, simplicity of surface tailoring, and controlled drug release properties . AlthougThe need for nontoxic and biocompatible AuNPs with small particle size, aggregation prevention, and the ability to introduce functionality to the particle surface for the conjugation of biomolecules has thus been identified. The search is currently underway for a one-step solution to all of these issues. The utilization of natural materials such as natural polysaccharides and gums might help to overcome this constraint.l-rhamnopyranosyl, β-d-glucuronopyranosyl, and 4-O-methyl-β-d-glucuronopyranosyl, α-l-arabinofuranosyl units , trypsin, EDTA, and phosphate-buffered saline (PBS) were purchased from Sigma Chemicals. Fetal bovine serum (FBS) was purchased from Gibco. Flasks  was powdered and filtered through a sieve with a mesh size of 250. A combination of Milli-Q water and ethanol in a 1:1 ratio was used to make a stock solution of 1% 4·3H2O) was dissolved in 0.25 mL Milli-Q water and 0.25 mL ethanol. It was kept for solubilization using a magnetic stirrer for 15 min.To obtain the gold chloride solution, 3.96 mg of gold chloride (HAuCl4 solution (10 mM) was added to 5 mL of GA solution (1% w/v). Then, the temperature was maintained at 80 ± 1 °C with continual stirring for an additional 30 min. One hour after the start of the experiment, a dramatic shift in color from colorless to ruby red could be noticed. This served as proof of the synthesis of AuNPs.Stabilized colloidal AuNPs were synthesized according to a previously described method, with slide modification . For synThe synthesized GA-AuNPs were confirmed by UV–Vis spectroscopy with slight modification to the method given by Nakamura et al. . Three mLTZ was slightly soluble in ethanol. To make it completely soluble, we added DI water and ethanol at a 1:1 ratio. The solution was stirred for 15–20 min. After stirring for 20 min, 15 mL of GA-AuNPs was added to this solution with continuous stirring at room temperature for 24 h. No precipitation of the drug was observed. It was further centrifuged at 15,000 rpm for 30 min. LTZ-encapsulated GA stabilized colloidal gold nanoparticles were collected for further study. The drug content in the supernatant was determined using UV–Vis absorption spectroscopy  at 240 nm. The % drug capping was established by Equation (1):Dynamic light scattering (DLS) was applied for size determination. Malvern Zetasizer Nano ZS  was used to measure the mean diameter, polydispersity index (PDI), and zeta potential (ZP) of LTZ-GA-AuNPs. The hydrodynamic diameter was evaluated after suspending the prepared colloidal nanoparticles in deionized water. The extent of DLS was prepared through a laser wavelength of 633.0 nm at 25 °C by a revealing angle of 90°. All samples were analyzed in triplicate and the three cycles were averaged.−1.Individual samples of either LTZ or LTZ-GA-AuNPs were combined with KBr and then compressed into a pellet using a hydraulic press. The resulting pellets were examined using an FTIR spectrophotometer  in the range of 4000–400 cmField emission scanning electron microscopy (FE-SEM)  was used for observing the morphology of the LTZ-GA-AuNPs. It was equipped with a tungsten filament, which covers the sample, gives high-quality pictures with minute electrical impulses, and has an advantage over high-resolution transmission electron microscopy (HR-TEM).HR-TEM and selected area electron diffraction (SAED)  were used for the morphology study. For this, a drop of the sample was drop cast on a carbon-coated copper grid. It was then air-dried at room temperature and stained with 2% uranyl acetate.As previously reported, in vitro LTZ release from LTZ-GA-AuNPs was evaluated using the dialysis bag technique in phosphate-buffered saline  and simulated cancerous conditions (pH 5.0) in vitro for 72 h, at 37 °C, respectively. The dialysis membrane used has a molecular weight cut-off (MWCO) between 12,000 to 14,000 Da. A sample of LTZ-GA-AuNPs equivalent to 5 mg of LTZ was dissolved in 5 mL of distilled water and sealed in a dialysis bag. Five hundred milliliters of either PBS pH 7.4 or sodium acetate buffer pH 5.0 was used as a release medium. Samples of 5 mL were withdrawn at regular time intervals for 72 h and replaced with an equivalent volume of fresh medium. The samples were quantified for the released drug by UV spectrophotometer at 240.00 nm.At is the absorbance of the treated supernatant, Ac is the absorbance of the negative control, and Ax is the absorbance of the positive control [The use of blood hemolysis tests determined the biocompatibility of the nanocarriers. A minor modification to the previously published procedure technique was performed . LTZ-GA- control .(2)% He® 96-well plates, Merck) at a 2 × 106 cells/well concentration. Different concentrations of LTZ and LTZ-GA-AuNPs were added to the wells seeded with cells. The plates were incubated for 48 h. Consequently, the sample-laden medium was replaced with 100 μL of fresh culture medium and 20 μL of MTT solution (5 mg/mL in PBS) in each corresponding well. An aliquot (85 μL) from the wells was removed, and 50 μL of DMSO from each well was added and wholly mixed with the pipette and further incubated at 37 °C for 10 min. From the microplate reader , cell viability was determined at 540 nm, which is based on the ability of viable cells to decrease the tetrazolium component of MTT into purple-colored formazan crystals [The cell viability in the Human breast cancer cell line (MCF-7) was determined by the MTT assay as previously described ,25. The crystals The % cell viability was calculated by Equation (3).50 value was determined by using a linear regression equation, i.e., y = mx + c.The ICp < 0.05 was considered statistically significant.Here, y = 50, m, and c values were derived from the viability graph. A value of A short-term physical stability revision was maintained to govern the influence of temperature on LTZ-GA-AuNPs. The stability of LTZ-GA-AuNPs was examined at temperatures of 25 ± 1 °C for 30 days. The mean particle size, polydispersity, and zeta potential of LTZ-GA-AuNPs were measured to assess physical stability .3+) to a neutral gold atom (Au0). GA was used as a reducing and capping agent in this experiment. Many variables, including temperature, reaction time, gum concentration, gold concentration, and the gold-to-gum ratio, were observed to influence the synthesis of gold nanoparticles in the study. The overall synthetic method did not use any toxic chemical reagents. The color of the gold solution did not change until it reached a temperature of 50 °C. At 60 °C, the color of the solution changed from colorless to violet due to a further increase in temperature as shown in In the current work, we synthesized LTZ-encapsulated GA-AuNPs by reducing a charged gold atom  was used, as shown in us study . The secFollowing the preparation, LTZ @ GA-AuNPs were characterized for their morphological features. The characters of AuNPs depend on morphological features such as the appearance and crystal characteristics of nanoparticles. The morphological examination was carried out using field emission scanning electron microscopy (FE-SEM) and high-resolution transmission electron microscopy (HRTEM). FE-SEM of the air-dried LTZ @ GA-AuNPs mainly showed spherical in shape, as shown in Nanoparticle shape and surface morphology were also investigated by HR-TEM analysis. We performed dynamic light scattering (DLS) to quantify the size distributions, polydispersity index (PDI), and zeta sizer to further understand these characteristics. According to A prospective drug carrier needs to have a high loading capacity as well as structural integrity. As a result, it is critical to investigate the maximum drug loading as well as relative conditions. Therefore, the capping of LTZ by GA-AuNPs was tested to assess their potential use as nanovectors in this context. The drug capping was found to be 42%.LTZ was chosen as a cancer model drug for breast cancer. As shown in v/v) was utilized, and the absorbance value of Triton X-100 was regarded to signify total (100%) hemolysis. Triton is a nonionic surfactant that causes the rupture of the RBC membrane, resulting in the release of hemoglobin. −1 were consistent with the limits of less than 15%. Furthermore, as AuNPs are biocompatible and non-toxic, they are considered to be superior drug delivery vehicles. The above studies on RBCs suggest that the synthesized LTZ-GA-AuNPs in the present study are potentially biocompatible and safely used inside the body.Hemolytic toxicity of GA stabilized gold nanoparticles was examined against RBC suspension (2% hematocrit). As a standard hemolytic agent, Triton X-100  were observed to be negligible. Based on the stability study, it can be concluded that the newly synthesized formulation is stable and can direct the exploration of applications in targeted drug delivery therapy.The novel LTZ @ GA-AuNPs formulation was studied for its stability for one month at room temperature (RT) as shown in Breast cancer in postmenopausal women is one of the leading causes of cancer-related deaths in women. The recent promising applicability of Letrozole (LTZ) as an anticancer drug has been approved for breast cancer treatment in postmenopausal women. However, this therapy has appreciable side effects. Nanoscale carriers, such as metal nanoparticles and polysaccharides, are being widely utilized as drug delivery vehicles to target tumor cells precisely. Biologically inspired green synthesis of nanoparticles has become a promising alternative against the chemically fabricated process considering reduced toxicity. This work introduced a new approach for breast cancer therapy using LTZ-GA-AuNPs as LTZ delivery. This study reports a safe and rapid method for the green synthesis and characterization of GA-AuNPs. The structural and morphological properties are confirmed by SEM and HR-TEM, respectively. Material characterizations confirmed the uniform LTZ within GA-AuNPs in the size range of 81.81 nm, 0.286 of PDI, and −14.6 mV zeta potential. The in vitro cytotoxicity with LTZ-GA-AuNPs showed better therapeutic efficacy than bare LTZ in the MCF-7 cell line. Thus, the LTZ-GA-AuNPs-based drug delivery system provided a reliable therapeutic strategy for treating breast cancer.


Eosinophilic chronic rhinosinusitis (ECRS) is a subtype of chronic rhinosinusitis (CRS) and is a refractory or intractable disease. However, a reliable clinical marker or an effective treatment strategy has not yet been established. ECRS is accompanied by excessive eosinophil infiltration and Th2 inflammatory response, which is closely related to tissue remodeling in the upper airways.We sought to investigate the effect of eosinophils on tissue remodeling in ECRS. The purpose of this study was to identify the effects of eosinophils on the expression of pro-inflammatory mediators and extracellular matrix (ECM) in nasal fibroblasts and the key mediators that stimulate them.Butyric acid was used to differentiate EOL-1 cells into eosinophils. We co-cultured differentiated EOL-1 cells and fibroblasts to measure the expression of pro-inflammatory mediators and ECM in fibroblasts. Among the cytokines secreted from the differentiated EOL-1 cells, factors that induced tissue remodeling of fibroblasts were identified.Treatment with butyric acid (BA) differentiated EOL-1 cells into eosinophils. Differentiated EOL-1 cells induced fibroblasts to produce pro-inflammatory mediators, IL-6 and IL-8, and tissue remodeling factor, VEGF. It also induced myofibroblast differentiation and overexpression of ECM components. Differentiated EOL-1 cells overexpressed osteopontin (OPN), and recombinant OPN increased the expression of IL-6, IL-8, VEGF, and ECM components in nasal fibroblast. OPN was overexpressed in the nasal tissue of patients with ECRS and was associated with the severity of CRS.Eosinophil-derived OPN stimulated nasal fibroblasts and contributed to inflammation and tissue remodeling in ECRS. Moreover, the expression level of OPN was proportional to the severity of ECRS. Therefore, OPN regulation is a potential treatment for ECRS. Chronic rhinosinusitis (CRS) is a multifactorial inflammatory disease involving the nose and paranasal sinus. The phenotype of CRS is heterogeneous and is classified into CRS with nasal polyp (CRSwNP) and CRS without nasal polyp (CRSsNP) depending on the presence or absence of nasal polyp. However, the pathogenesis of the disease is difficult to determine from this classification. Hence, providing customized treatment becomes challenging as it is difficult to distinguish the immune response, disease progression, and histopathological differences in patients –3. ThereECRS is mainly observed in western countries. In ECRS, Type 2 inflammation is dominant along with tissue eosinophilia and is difficult to treat. Tissue eosinophilia is highly involved with recalcitrant nature of the disease and nasal polyp recurrence rate after endoscope sinus surgery. The tissues of patients with ECRS mainly show edema. ECRS is more severe than non-ECRS, with higher rates of recurrence. In contrast, non-ECRS is more prevalent in Asia. Glandular hyperplasia and dense collagen accumulation are observed among Asian patients , 6. Kim Eosinophils are one of the granular innate immune cells and play a key role in type 2 inflammation. Eosinophils differentiate into a mature form, mediated by IL-5, and infiltrate into the tissues, inducing type 2 inflammation . Tissue in vitro model for eosinophil study. EOL-1 cell can be differentiated into eosinophil by butyric acid (BA). In previous studies, it was found that BA treatment of EOL-1 increased the expression of eosinophil markers such as CCR3, IL-5Ra, and eosinophil secondary granules  and differentiated them into eosinophil-like cells. It was also reported that eosinophil TLR receptors  were expressed in differentiated EOL-1 cells. In in vitro studies, primary eosinophils have disadvantages such as low survival rate and difficult long-term co-culture, so the EOL-1 cell line is often used as an alternative cell (EOL-1 is a human eosinophilic cell line and represents a useful ive cell –15.via repeated insults to the tissue (Fibroblasts are the primary cells to synthesize extracellular matrix (ECM), collagen, and stroma , and alse tissue .Therefore, in this study, we first aimed to evaluate the effects of eosinophils on tissue remodeling, and inflammatory properties of fibroblasts. Second, we tried to identify the key mediators involved in the eosinophil-mediated stimulation of fibroblasts. Finally, the effect of the eosinophil-derived mediator on the development and severity of CRS was confirmed.Uncinate process samples from the nasal cavity, and polyp tissues were obtained from 25 patients . Normal uncinate process tissues were harvested during rhinoplastic surgery. Nasal polyp tissues were obtained from the region of the middle meatus at the beginning of the endoscopic surgical procedure of CRS patients. The CRS was diagnosed based on historical, endoscopic, and radiographic criteria and CT findings of sinuses according to the 2012 European position paper on rhinosinusitis and nasal polyps (EPOS) guidelines. All subjects were recruited from the Department of Otorhinolaryngology, Korea University Medical Center, Korea. All tissues were derived from patients with no signs of inflammation, allergies, asthma, or aspirin sensitivity. No patients had a history of consuming oral steroids, non-steroidal anti-inflammatory drugs, antihistamines, or antibiotics for at least past 2 months. Informed consent was collected according to the Declaration of Helsinki. This study was approved by the Korea University Medical Center Institutional Review Board, which also authorized the research. It was carried out in accordance with the guidelines of the Human Ethics Committee of the Korea University Guro Hospital (2020GR0308). Clinical characteristics of patients are summarized in GSEA was performed to determine whether inflammation and ECM regulation are involved in CRS. GSEA software v4.1.0 was used to analyze the NGS data and GSE36830 data. Gene sets were analyzed using 10 hallmarks involving the inflammatory response and NABA ECM regulators. The number of permutations was set as 1000, the type was gene_set, and the chip platform was human_NCBI_Gene_ID_MSigDB.v7.4.chip.5 cells in a 100mm culture dish, and 3x105 cells in a 60mm culture dish.Nasal fibroblasts were obtained from the inferior turbinates of patients who underwent rhinoplasty. Inferior turbinate tissues were subjected to enzymatic digestion with collagenase , hyaluronidase , and DNase  to obtain fibroblasts. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) , 10,000 μg/mL streptomycin (Invitrogen), and 1% 10,000 U/mL penicillin (Sigma). The purity of the obtained nasal fibroblasts was confirmed by characteristic spindle-shaped cell morphology and flow cytometry. Fibroblasts were seeded with 7x106 cells in a 100mm culture dish, and 5x105 cells in a 60mm culture dish.EOL-1 cells were purchased from Sigma and cultured in RPMI-1640 media supplemented with 10% heat-inactivated FBS , 10,000 μg/mL streptomycin (Invitrogen), and 1% 10,000 U/mL penicillin (Sigma). EOL-1 were seeded with 1x105 cells, and EOL-1 were seeded with 6x105 and 1.2x106, respectively. In addition, indirect culture was performed using a transwell. Differentiated EOL-1 cells were cultured in the upper chamber, and the fibroblasts were cultured in the lower chamber.EOL-1 cells were cultured in RPMI-1640 media containing 10% FBS and treated with 0.5 mM butyric acid (BA) to differentiate them into eosinophils. Differentiated cells were co-cultured with fibroblasts in the DMEM medium containing 10% FBS at a ratio of 1:2 and 1:4. In 60mm culture dish, fibroblasts were seeded with 3x10® Cytotoxicity Assay  was performed. Briefly, EOL-1 cells were seeded into a 96-well plate at a density of 2 x 104 cells per well. After seeding, the medium was replaced with butyric acid and treated with IncuCyte® Cytotox Green Reagent (Essen BioScience Inc.). The viability of the EOL-1 cells cultured at 37˚C in an atmosphere containing 5% CO2 and 95% humidity was measured at 72 h. Apoptotic EOL-1 cells were stained by IncuCyte® Cytotox Green Reagent and the viability of EOL-1 cells was evaluated by measuring green fluorescence through IncuCyte® software (Essen BioScience Inc.).To determine the cytotoxicity of butyric acid, IncuCyteTo evaluate the cytotoxicity of butyric acid, WST-1 assay was performed. EOL-1 cells were seeded into a 96-well plate and 50 µL WST-1 reagent was added per well. The cells were incubated for 4 h and the results were obtained by measuring the absorbance using a spectrophotometer at 420-480 nM.-ΔΔCT method. Each experiment was repeated at least three times, and GAPDH was used as an internal control.Total RNA was extracted using the Trizol reagent (Invitrogen). The cDNA was synthesized using the Maxime RT PreMix kit  according to the manufacturer’s protocol. Amplification reaction was performed with the following steps: an initial 2 min denaturation step at 94°C; 40 cycles of 94°C for 5 seconds, 60°C for 10 seconds, 72°C for 20 seconds. All reactions were performed in 20 μL volume. Real-time PCR was performed on the QuantStudio 3 system  with 100 ng cDNA template, 400 nM of each primer, and 10 mL Power SYBR Green PCR Master Mix (Applied Biosystems) in 20 μL. Analysis of relative gene expression was conducted using the 26 cells in a 100mm culture dish and cells were treated with or without 0.5 mM BA and cells were harvested after 3-5 days. Cells were incubated with CCR3-PE  and IL-5Ra-FITC  conjugated antibodies for 30 minutes at 4°C and analyzed by FACS. All groups were analyzed with 10,000 cells. Flow cytometry analysis was performed using a Flow Cytometer Analyzer (LSR Fortessa X-20).Flow cytometry was performed to confirm that EOL-1 cells differentiated into eosinophils. EOL-1 were seeded as 1x10To evaluate the protein expression and localization, immunocytochemical staining was performed. Before using the cell culture slides, they were coated with poly-L-lysine for 4 h, after which they were exposed to UV light for 6 h. Fibroblasts and EOL-1 cells were seeded on the poly-L-lysine coated slides. Subsequently, cells were fixed with 4% paraformaldehyde and permeabilized with 0.01% Triton X-100 in 1% bovine serum albumin for 10 minutes. Then, cells were blocked with 3% bovine serum albumin for 1 h. Fibroblasts were incubated with primary antibodies; anti-Fibronectin (1:200), anti-SMA (1:200), anti-collagen type I (1:200). Eol-1 were incubated with primary antibodies; anti-CCR3 (1:200), anti-IL-5Ra (1:200) and then incubated with secondary antibodies; anti-mouse Alexa 488 (Invitrogen) or anti-rabbit Alexa 555 (Invitrogen) secondary antibodies. Finally, cells were counterstained with 4’-6-diamidino-2-phenylindole for 10 min and stained cells were visualized using a confocal laser scanning microscope .Nasal polyp and uncinate process tissue sections were deparaffinized and rehydrated. Sections were stained with primary antibodies, anti-OPN  and anti-IL5Ra  overnight at 4°C. Subsequently, they were incubated with anti-mouse Alexa 488 (Invitrogen) or anti-rabbit Alexa 555 (Invitrogen) secondary antibodies or 3,3’- diaminobenzidine (DAB) substrate (Zytomed systems). Sections were counterstained with hematoxylin or 4’-6-diamidino-2-phenylindole (Sigma-Aldrich).5 cells, and EOL-1 were seeded with 6x105 and 1.2x106, respectively. After 3 days of co-culture, the levels of IL-6, IL-8, and VEGF were measured using ELISA . In the second experiment, the expression levels of IL-6, IL-8 and VEGF were checked after 3 days of treatment with OPN (0.5-4ug/ml) in fibroblasts. The sample of ELISA was carried out using the supernatant from which cells were removed.First, co-culture of fibroblasts and EOL-1 was performed. In 60mm culture dish, fibroblasts were seeded with 3x10Briefly, each well was blocked with blocking buffer for 2 h and washed using a wash buffer. Antibodies against IL-6, IL-8, or VEGF were added to the media and incubated for 2 h. A substrate solution and stop solution were added sequentially, and the optical density of each well was determined within 30 minutes using a microplate reader (Bio-Rad).A cytokine and chemokine array  was used to survey the changes in the expression levels of 102 cytokines and chemokines in EOL-1 cells after treatment with or without BA. Cell culture supernatant was collected, diluted, and incubated overnight with the contents of the array kit. The membrane was washed to remove unbound material followed by incubation with a cocktail of biotinylated detection antibodies. The membrane was incubated with streptavidin-horse radish peroxidase and chemiluminescent detection reagents, and a signal was generated at each capture spot corresponding to the amount of bound protein. The membrane was exposed to X-ray film for 10 minutes, and profiles of mean spot pixel density were analyzed using Quantity One software (Bio-Rad).3 pieces, washed three times with phosphate-buffered saline, and cultured in DMEM supplemented with 2% FBS (Invitrogen) 1% 10,000 units/mL penicillin, and 10,000 μg/mL streptomycin (Invitrogen).Nasal inferior turbinate and polyp tissues were cut into 2 to 3 mmt-test or one-way analysis of variance followed by Tukey’s test . Significance was established at a 95% confidence level and P < 0.05 indicated statistical significance.Results were obtained from at least three independent experiments. Statistical significance of differences between control and experimental data was analyzed using the unpaired We performed GSEA to determine the degree of inflammation and the amount of ECM produced in ECRS along with the effect of eosinophil on these aspects. The gene set database used Hallmark_inflammatory_response and NABA_ECM_Regulators. We observed that the correlation between inflammation and the expression of ECM regulators was higher in the CRS patient group than in the healthy control group, and a significant correlation was also found with the ECRS patient group. Among them, genes such as those encoding pro-inflammatory mediators IL-6 and IL-8 and ECM markers such as a-SMA and fibronectin showed a high normalized enrichment score (NES).  of fibroblasts were partially elevated. a-SMA and collagen type 1 showed the greatest increase in RNA levels under co-culture conditions with differentiated EOL-1. However, Fibronectin did not show a significant increase in gene expression  increased 11-fold, 6.7-fold, and 5.2-fold, respectively, in EOL-1 cells differentiated with butyric acid compared to those in undifferentiated EOL-1 cells  , a straight-chain fatty acid, can differentiate EOL-1 cells into mature eosinophil-like cells . Using fWe used a cytokine array to identify the components of EOL-1 cells that mediated cytokine secretion and ECM production by fibroblasts. EOL-1 cells differentiated with butyric acid showed differential cytokine expression pattern compared to EOL-1 cells cultured under normal conditions. In particular, the expression of OPN, Plasminogen activator inhibitor-1 (PAI-1), and sex hormone-binding receptor (SHBR) in differentiated EOL-1 cells increased 11-fold, 6.7-fold, and 5.2-fold, respectively, and the expressions of thymus and activation-regulated chemokine (TARC) and macrophage inhibitory cytokine 1 (MIC-1) were also slightly elevated. Among them, OPN showed stimulatory effects on fibroblasts comparable to those observed with co-culture experiments. OPN, also known as T lymphocyte activation 1 and secreted phosphoprotein 1, is a highly negatively charged ECM protein. It was discovered in the bone as an ECM protein . HoweverOPN expression was increased in both CRSsNP and CRSwNP patients, and among them, the eosinophilic type of CRSwNP was most frequently expressed in the nasal polyp tissues of patients. The protein levels of OPN were significantly higher in eosinophilic NP tissues than in the non‐eosinophilic NP tissues. In addition, IHC double staining results confirmed that the eosinophils in the eosinophilic NP tissue were OPN+ cells.Eosinophilic CRS is a relatively dominant type of eosinophil in the infiltrated immune cells in mucosa tissue. In fact, they show higher levels of inflammation and higher severity than non-eosinophilic ones. Therefore, the probability of recurrence after surgery is high and the prognosis is poor. Inflammatory response of nasal tissue caused by foreign antigen increases vascular permeability in the tissue and increases the residual eosinophil in the tissue. In this study, it was confirmed that these EOL-1 can secrete OPN. OPN has a chemotaxis effect that can move eosinophils into tissues, so it may accelerate tissue inflammation and tissue eosinophilia through positive feedback . The abiBased on the finding that eosinophilic markers correlated with tissue remodeling markers in CRS patients, we investigated the effects of eosinophils on the fibroblasts of the upper airway along with identifying the factors secreted from eosinophils involved in this process. We confirmed that EOL-1 cells differentiated by BA increased the expression of mediators related to inflammation and tissue remodeling under three different co-culture methods. To identify the factors secreted by EOL-1 cells that stimulated the fibroblast to produce pro-inflammatory cytokines and mediated their differentiation into the active form, we used a cytokine array. Among the cytokines released from EOL-1 cells activated by BA, treatment with OPN alone showed similar effects on fibroblasts compared to the effects observed in co-culture experiments. Moreover, the expression level of OPN in tissues from CRS patients correlated with the level of eosinophil markers. Taking these results together, the level of OPN expression in tissues can be used as a diagnostic marker for ECRS. In addition, regulation of OPN expression could be a potential therapeutic strategy for ECRS.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.This study was approved by the Korea University Medical Center Institutional Review Board, which also authorized the research. It was carried out in accordance with the guidelines of the Human Ethics Committee of the Korea University Guro Hospital (2020GR0308). The patients/participants provided their written informed consent to participate in this study.H-WY conceived the study, designed and performed the experiments, analyzed the data, and wrote the manuscript. J-HP, M-S, J-MS, and DK confirmed the data and discussed this study. I-HP supervised the research and reviewed the manuscript. All authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.This research was supported by a Korea University Grant .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In this work, we studied the combination of nitrogen-doped carbon quantum dots (N-CQD), a hydroxide-ion conducting ionomer based on polysulfone (PSU) and polyaniline (PANI), to explore the complementary properties of these materials in high-performance nanostructured electrodes for the oxygen reduction reaction (ORR) in alkaline solution. N-CQD were made by hydrothermal synthesis from glucosamine hydrochloride (GAH) or glucosamine hydrochloride and N-Octylamine (GAH-Oct), and PSU were quaternized with trimethylamine (PSU-TMA). The nanocomposite electrodes were prepared on carbon paper by drop-casting. Furthermore, we succeeded in preparing PSU-TMA + PANI + GAH-Oct fibers by electrospinning. The capacitance of the electrodes was investigated by cyclic voltammetry and impedance spectroscopy, which gave similar trends. The ORR was investigated by linear sweep voltammetry at rotating disk electrode speeds between 250 and 2000 rpm in an oxygen-saturated 1 M KOH solution. Koutecky–Levich plots showed that four electrons were exchanged for nanocomposite electrodes containing CQD. The highest reduction currents were measured for the electrodes containing GAH-Oct. The Tafel plots gave the lowest slope and the most positive half-wave potential for PSU-TMA + PANI + GAH-Oct fibers. The best electrocatalytic activity of this electrode could be related to the high amount of graphitic nitrogen in GAH-Oct. Long-term cycling tests showed no significant modification of the onset potential, but a change of the current in the mass transport limited region, indicated the evolution of the microstructure of the nanocomposite ORR electrode modifying the mass transport conditions during the first 400 cycles before reaching stationary conditions. FTIR spectra were used to study possible electrode degradation after the ORR in 1 M KOH: the only change was due to the reaction of PANI emeraldine salt to emeraldine base, whereas the other constituents of the multiphase electrode did not show any degradation. It is wtabolism ,7,8. Thetabolism ,12,13,142O/H2O2 selectivity are further influenced by oxygen-containing moieties [2 adsorption and are efficient mediators of the two-electron reduction of O2 to HO2− and the subsequent reduction to HO− [Substitutional N-doping is a powerful method to improve graphitic carbon materials for various applications, including the catalysis of important chemical reactions, such as the ORR ,16,17,18moieties , such asn to HO− :(2)O2+2 − ions near the catalytically active centers. Polyaniline (PANI), that reported to present interesting catalytic properties for the ORR, was added in some electrodes to verify if it boosts the catalytic performance [We have recently reported the advanced synthesis of CQD with average sizes in the nanometer range by pyrolysis, microwave irradiation and hydrothermal treatment. The content and position of N in the CQD can be tailored by functionalization with various nitrogen-containing compounds and the synthesis method and conditions . CQD preformance ,23,24. TThe X-ray diffractogram of a PSU-TMA + PANI + GAH-Oct multiphase electrode on carbon paper is shown in The double-layer capacitance C is determined from the non-Faradaic current density at various scan rates, according to Equation (4):j vs. the scan rate dU/dt are presented in C, determined from the slope of the straight lines according to Equation (4), is 130 μF and the consistency of the values is good. Plots for other electrodes are shown in Typical linear plots of the current density The capacitance of carbon paper is much lower than that of any nanocomposite electrode, which shows high electrochemically active surface areas (EASA). It may be noted that the fiber electrode does not have the highest EASA, probably because some agglomeration takes place during the preparation . Drop-casting is a valid method to provide high surface area electrodes.2. Assuming a double layer capacitance value of 20 μF/cm2 for flat electrodes [2. However, lower double-layer capacitance values were reported for carbon-based materials [Dividing the capacitance by the geometrical electrode area gives values between 1.8 and 6.1 mF/cmectrodes , the EASaterials .Typical impedance spectra of various ORR electrodes are shown in i is the imaginary unit, ω the angular frequency, Y the CPE value, and m the CPE exponent, which is characteristic of the physical nature of the element: a value near 1 indicates an imperfect capacitance [R, Y, and m are reported in m = 0.95), the Y value is consistent with the double layer capacitance determined by CV measurements  and a constant phase element (CPE) Q. The impedance of the CPE can be written:acitance . The besurements . The Y dexponent . The two2- and O2-saturated 1 M KOH solution. A well-defined reduction peak is observed with an onset potential around −0.2 V vs. Ag/AgCl in the O2-saturated, but not in the N2-saturated solution, showing that O2 is reduced on the electrode with PSU-TMA + PANI + GAH-Oct.− transport.A comparison of LSV of PSU-TMA + PANI + GAH-Oct electrodes with commercial Pt/C foil is shown in 2 as indicated in The electrocatalytic activity of N-doped carbon materials is attributed to the capacity of the nitrogen to induce polarization on the neighboring carbon, the active center for the chemisorption of OThe presented results show that ORR electrodes containing GAH-Oct have a higher catalytic activity than that with GAH, although the latter electrode has a higher electrochemically surface area. A convincing interpretation can be based on the position of nitrogen atoms inside the CQD and their influence on the ORR electrocatalytic activity. It was demonstrated previously that graphitic nitrogen has a distinctly higher catalytic activity than pyridinic or pyrrolic nitrogen . We haveLinear scan voltammograms (LSV) at various rotational speeds of the RDE are shown in n of transferred electrons per oxygen molecule can be determined using the Koutecky–Levich equation between the current j and the rate of the rotating-disk electrode ω [The number ctrode ω ,34:(6)1jkj is the kinetic current and B is the Levich slope, which is given by the relation:In this equation, A is the geometrical electrode area, F the Faraday constant , c(O2) the saturation concentration of oxygen in 1 M KOH (8 × 10−7 mol cm−3 [D(O2) the oxygen diffusion coefficient (1.5 × 10−5 cm2 s−1 in 1 M KOH [ν the kinetic viscosity of the 1 M KOH solution (0.0094 cm2 s−1) [Here, mol cm−3 ,36), D(O 1 M KOH ), and ν cm2 s−1) . Typical−1 s−1/2). The number of exchanged electrons is reported in n < 3, so that more hydrogen peroxide than water is formed by the ORR.The slopes of Koutecky–Levich plots for nanocomposite electrodes containing GAH and GAH-Oct are in good agreement with the value for 4 transferred electrons per oxygen molecule (56 mAη| and the current density |j| [Tafel plots for carbon paper and electrodes with GAH-Oct are shown in sity |j| :(8)|η|=2R is the ideal gas constant (R = 8.314 J K−1 mol−1), T the absolute temperature, α the symmetry coefficient, assumed to be 0.5, and j° the exchange current density. The slope for carbon paper is in the order of 90 mV/decade, intermediate between values reported in the literature for low current densities (2.3·RT/F) indicating a pseudo-two-electron-process to be the rate-determining step [RT/0.5·F) [In this equation, ing step ,36, and T/0.5·F) , where tT/0.5·F) ,39.The lower Tafel slopes with GAH-Oct indicate a better catalytic activity. The lowest value is observed for PSU-TMA + PANI + GAH-Oct fibers, which present also the most positive half-wave potential , confirmA cycling test for the PSU-TMA + PANI + GAH-Oct electrode is shown in FTIR spectra of PANI-GAH-Oct and PSU-TMA + PANI + GAH-Oct, recorded before and after the electrocatalytic experiments, are presented in −1 originate from the C-H stretch of octyl groups attached to CQD. The main difference in the spectra is the shift to higher wavenumber of the peak due to the stretching vibration of the nitrogen quinoid ring, which is responsible for the catalytic activity of PANI [−1 for EB, and 1565 cm−1 for ES [−1 in EB, and only as a shoulder at 1480 cm−1 in ES. The broad peak at 1400 cm−1, which is observed only in the initial sample, can be attributed to the asymmetric stretching of carbonate groups, the counter-ion of ammonium groups initially present in GAH-Oct. The peak disappears after treatment in basic solution [−1 for EB, and 1298 cm−1 for ES. From the out-of-plane C-H bonding region, it is possible to estimate the substitution pattern of the aromatic rings, resulting in a peak at 820 and 800 cm−1 for EB and ES respectively, corresponding to 1,4-substitution. The same behavior is also observed in the spectra of GAH-Oct alone, presented in the PANI-ES is neutralized to PANI-EB after immersion in the alkaline solution used for the ORR b. The fo of PANI ,23,24, p1 for ES ,41. The solution . The C-N−1 the C-H stretching due to the overlapping vibrations of methyl groups in polysulfone and trimethylammonium, and the aliphatic chain of GAH-Oct. At 1250 cm−1 the vibration of the ether link of PSU is visible [−1 is shifted to 1580 cm−1 in ES. An intense peak appears after the ORR at 1380–1400 cm−1, attributed to carbonatation of PSU-TMA . In conclusion, the constituents of the nanocomposite electrode do not degrade during the ORR.More complex are the spectra of PSU-TMA + PANI + GAH-Oct electrodes b, becaus visible . As obsePolysulfone was purchased from Solvay . Polyaniline , D-(+)-glucosamine hydrochloride (GA), N-octylamine (Oct), N,N, N-trimethylamine , N-methylpyrrolidone (NMP), dimethylformamide (DMF) and the other chemicals were reagent-grade and were used as received from Sigma-Aldrich. Carbon paper (AvCarb EP55) and Pt/C foil were obtained from Fuel Cell Store.GAH and GAH-Oct2O or in 1 mL H2O and 0.4 mL N-octylamine (2.1 mmol) for GAH and GAH-Oct, respectively. The mixtures were stirred for 40–50 min at 50 °C to obtain uniform solutions, then transferred in an autoclave and inserted into an oven at 190 °C for 12 h. The autoclave was then cooled down to room temperature and the solid was dispersed in deionized water for GAH and chloroform for GAH-Oct, which is hydrophobic. The final solutions were centrifugated. The solids were used for the electrode preparation. The characterization of GAH and GAH-Oct and transmission electron microscopy images are reported in [The synthesis of GAH and GAH-Oct by a hydrothermal treatment was previously presented . In brieorted in .PSU-TMA4 in chloroform. After purification, the degree of chloromethylation was 1.2. PSU-TMA was obtained by reacting PSU-CM with TMA in DMSO. The resulting degree of amination was 0.9 and the ion exchange capacity (IEC) was 1.8 meq/g.The anion exchange ionomer was prepared by the chloromethylation route followed by amination as described in . ChloromThe chemical structures of PSU-TMA, PANI , and CQD, are represented in The electrodes were made by coating carbon paper with 5 μL of a suspension of the constituents using a microsyringe. The coated electrodes were dried at 80 °C for 24 h. The preparation of the suspensions is described below.PANI + GAH-OctAn amount of 0.15 g of finely powdered GAH-Oct was dispersed in 0.60 g of DMF under vigorous stirring. Later, 0.07 g of PANI was added to the above solution.PSU-TMA + PANI + GAHAn amount of 0.45 g of PSU-TMA in 2.0 g of DMSO was inserted into a small, closed glass vial with 0.07 g of PANI, and 0.15 g of finely powdered GAH was added with 0.60 g of DMF as solvent.PSU-TMA + PANI + GAH-OctThe same concentration of PSU-TMA and PANI in DMSO was maintained and 0.15 g GAH-Oct with 0.68 g NMP was added to disperse the materials well. In all cases, the final solutions were kept overnight under stirring before drop-casting the electrodes.Furthermore, fibers of PSU-TMA + PANI + GAH-Oct were obtained by electrospinning using a Starter Kit-Random (Linari Engineering). A 10 mL syringe with a 0.8 mm diameter needle was mounted on the syringe pump setting a flow rate of 0.04 mL/h. The fibers were electrospun at an applied voltage of 15 kV with a needle/collector (carbon paper) distance of 16 cm. SEM micrographs  of a typical electrospun electrode containing PSU-TMA, PANI and GAH-Oct are shown in Despite much effort, similar attempts to prepare electrospun fibers with GAH or without PANI were, unfortunately, unsuccessful.X-ray diffractograms were recorded using a Bruker D5000 diffractometer with Cu Kα radiation , 0.04° steps, and 2 s step time. −1 with a resolution of 0.5 cm−1.FTIR spectra were recorded using a Perkin Elmer Spectrum Two spectrometer  equipped with an ATR crystal diamond module in a wavenumber range of 500–4000 cmCyclic voltammetry (CV), linear sweep voltammetry (LSV), and electrochemical impedance spectroscopy (EIS) measurements were performed using a Biologic VMP3 potentiostat with a three-electrode cell. 2 area  was used as the working electrode. The coated carbon paper samples were fixed on the glassy carbon disk. A 4 cm2 platinum foil was the counter-electrode and saturated Ag/AgCl served as reference electrode (E = 0.197 V vs. NHE). All experiments were conducted at room temperature. The O2- and N2-saturated 1 M aqueous KOH solutions were obtained by bubbling the pure gases for at least 40 min through the liquid. A glassy carbon rotating disk electrode with 0.07 cmThe scan rate for the CV and the LSV measurements was 20 mV/s with various rotating speeds of the RDE between 250 and 2000 rpm. The long-term cycling tests were performed between −0.2 and −0.7 V vs. Ag/AgCl at 1500 rpm with a 50 mV/s scan rate. The impedance spectra were recorded using a Biologic VMP3 with an a.c. amplitude of 20 mV in a frequency range between 1 Hz and 1 MHz.We studied the electrocatalytic properties for the ORR of nanocomposites combining a hydroxide ion-conducting ionomer (PSU-TMA), polyaniline (PANI), and CQD prepared by hydrothermal synthesis from glucosamine (GAH) without, and with, N-octylamine (GAH-Oct). Application of the Koutecky–Levich equation to the linear sweep voltammograms of the ORR showed a four-electron reduction for the catalytic electrodes containing CQD. The best electrocatalytic activity was found for PSU-TMA + PANI + GAH-Oct fibers. The lower Tafel slope and most positive half-wave potential of this nanocomposite electrode could be attributed to the high amount of graphitic nitrogen in the CQD that was previously shown to improve the electrocatalytic activity for the ORR.Long-term cycling experiments showed good stability of the onset potential, but the current decrease in the plateau region during the first 400 cycles showed a microstructural evolution of the nanocomposite electrode that modified the mass transport. The FTIR investigation of the nanocomposite electrodes before and after the ORR in alkaline solution mainly showed the neutralization of PANI emeraldine salt, but no significant degradation of the constituents of the multiphase electrodes.


In conclusion, this newly developed whole-organ tissue clearing and imaging system provides comprehensive information at the whole-mount scale and has great potential for kidney research. Our data suggest that renal sympathetic hyperinnervation is involved in HF associated with renal dysfunction.Developing a three-dimensional (3D) visualization of the kidney at the whole-mount scale is challenging. In the present study, we optimized mouse whole-mount kidney clearing, which improved the transparency ratio to over 90% based on organ-specific perfusion (OSP)-clear, unobstructed brain imaging cocktails and computational analysis (CUBIC). The optimized OSP-CUBIC-compatible 3D immunostaining and imaging simultaneously visualized the high-resolution 3D structure of the whole-mount renal microvascular, glomerulus, and accompanying wrapped traveling sympathetic nerves in mice. A mouse model of pressure overload-induced heart failure (HF) was then established by minimally invasive transverse aortic constriction (MTAC). Further 3D quantification revealed renal sympathetic hyperinnervation (6.80 ± 1.04%  Kidney dysfunction is among the most common coexisting conditions in patients with acute or chronic heart disease . The terThe conventional explanation for the development of CRS in the setting of cardiac disorders focuses on the inability of the failing heart to generate forward flow, thus resulting in prerenal hypoperfusion. Inadequate renal afferent flow activates the sympathetic nervous system (SNS), the renin–angiotensin–aldosterone system (RAAS) axis, and arginine vasopressin secretion, eventually leading to fluid retention, higher preload, and worsening pump failure . Howeverin situ-hybridization-compatible tissue hydrogel (CLARITY)]  imaging of intact tissues and even some entire organisms have been developed in recent years. Three major tissue-clearing approaches are currently available: hydrophobic, hydrophilic, and hydrogel-based . These tLARITY)]  provide LARITY)]  reagentsLARITY)] .To better understand renal abnormalities and regulation in the whole-mount kidney, we developed a method of whole-organ tissue clearing that is compatible with 3D immunostaining, 3D imaging, and analysis to show clear macro- and microstructures of the whole-mount mouse kidney. Sympathetic hyperinnervation of the kidney in pressure overload-induced HF was observed by applying an optimized whole-organ clearing and imaging protocol.Randomization: Each experimental mouse had a unique number that was generated by the “RAND” function in Microsoft Excel, and the mice were divided into the study groups in numerical order of the unique number.Blinding: All of the experimental data were verified by an independent investigator who was blinded to the study group.Number of replicates: All of the experiments included biological replicates. The number of samples in each experiment is described in each figure legend. For all of the experiments, control data were acquired concurrently with data in which statistical comparisons were performed.Male adult (8–12 week old) C57BL/6N mice were purchased from Beijing Vital River Laboratory. The mice were maintained on a 12/12 h light/dark cycle and fed with a normal laboratory rodent diet. All of the mice were maintained in an animal facility that was approved by the Association for the Assessment and Accreditation of Laboratory Animal Care at China-Japan Friendship Hospital. All of the animal experiments were approved by the Institutional Animal Care and Use Committee of China-Japan Friendship Hospital and complied with all relevant ethical regulations.N-butyldiethanolamine , and 5 wt% Triton X-100 ]. After perfusion with various reagents, the kidney was dissected from the mouse and immersed in 10 ml of 1/2 CUBIC-L  in a 50 ml tube overnight at 37°C with gentle shaking. The next day, 10–15 ml of CUBIC-L was replaced in the 50 ml tube and delipidated/decolorated for another 5 days at 37°C with gentle shaking. After tissue clearing, the sample was washed in PBS for 2 h (three times) at room temperature with gentle shaking. The washing tube was replaced after each washing step to completely remove Triton X-100. The sample was then used for 3D immunostaining, refractive index (RI) matching, or temporary storage. For temporary storage, the kidney was immersed in 10 ml of 40% (wt/vol) sucrose in PBS with shaking at room temperature overnight. When the samples sank to the bottom, they were placed in O.C.T. compound and immediately stored at −80°C.The organ-specific perfusion-CUBIC (OSP-CUBIC) clearing protocol was optimized based on the conventional CUBIC protocol . Briefly2O) for 2 h at 37°C with gentle shaking. The kidney was then removed to 500 μl staining buffer with 1:200 anti-Tyrosine hydroxylase (TH) antibody  for 7 days at 37°C with shaking. After washing with 1% PBST (1% Triton X−100), the kidney was incubated with 500 μl staining buffer with secondary antibody conjugated to Alexa Fluor 647  for 7 days at 37°C. After washing with 1% PBST for several hours, the samples were immersed in 1% paraformaldehyde overnight at room temperature, followed by another three cycles of PBS washing. The kidney was then placed in 5 ml of 1/2 CUBIC-R + (N)  in a 10 ml tube overnight at 37°C with gentle shaking. The next day, 5 ml of CUBIC-R + (N) was replaced in the 10 ml tube and incubated for another 4 days.For 3D immunostaining of whole-mount kidney sympathetic nerves, OSP-CUBIC-cleared kidneys were sufficiently washed with PBS solution to remove clearing reagents. The kidney was first immersed in 5 ml staining buffer  that was equipped with a white-light laser module (NKT SuperK Extreme EXW-12), six fixed light-sheet generating lenses, an sCMOS camera , and a 2 × objective lens (Olympus MVPLAPO) covered with a 6 mm working distance dipping cap. Sixteen-bit optical sectional images of two channels  were acquired with a total magnification of 1.26 (N.A. = 0.14) with a z-step of 5 μm. During image acquisition, the optically cleared sample was immersed in an RI-matched oil mixture of silicon oil HIVAC-F4  and mineral oil .The confocal images were acquired using a Leica TCS SP8 microscope  that was equipped with a 25 × objective (N.A. = 0.95), with a voxel size of 0.8 μm × 0.8 μm × 1.98 μm. Two color fluorescence images with excitation wavelengths of 561 and 640 nm were taken for imaging blood vessels and sympathetic nerves, respectively. The cleared kidney was immersed in the RI-matched solution during imaging as described above.To quantify the 3D immunofluorescence information, the fluorescent image stacks that were acquired using light-sheet fluorescence microscopy (LSFM) were processed as follows. Background subtraction was performed, and then the images were segmented using the surface module in Imaris 9.6  to calculate the TH signal-positive volume. To calculate kidney volume, the fluorescent images were segmented with the Li algorithm  in ImageMinimally invasive transverse aortic constriction (MTAC) was surgically induced as previously described . After tTransthoracic echocardiography was performed using a VisualSonics Vevo 1100 system that was equipped with an MS400 transducer . After 1 week, the previously experimental mouse was anesthetized with isoflurane. After depilating hair, the mouse was secured in the supine position on a platform. Ultrasound gel was then applied to the mouse’s chest. Using an MS400 probe and a high-frequency ultrasound system, color Doppler and pulsed-wave Doppler scanning were performed on the left and right carotid arteries and aortic arch. Blood flow of the left common carotid artery, right carotid artery, and constricted site was evaluated.Systolic function was measured 0, 1, 4, 6, and 8 weeks after MTAC surgery or sham surgery. Indices of systolic function were obtained from short-axis M-mode scans at the midventricular level, indicated by the presence of papillary muscles. During echocardiogram acquisition, heart rate was maintained at 450–550 beats per min. All parameters were measured at least three times, and means are presented.The kidneys were collected and fixed in 4% paraformaldehyde overnight and processed for routine paraffin histology [5 μm sections stained with hematoxylin and eosin (H&E) and Masson’s trichrome]. The renal fibrotic area was quantified in three microscopic fields per kidney using ImageJ 1.52n software .The levels of brain natriuretic peptide (BNP), creatinine (CRE), and blood urea nitrogen (BUN) in plasma samples were measured using the BNP enzyme-linked immunosorbent assay kit , CRE assay kit , and BUN assay kit  according to the manufacturer’s instructions.The level of urine albumin was measured using the mouse albumin enzyme-linked immunosorbent assay kit , according to the manufacturer’s instructions. Twenty-four-hour mouse urine was collected with mouse metabolic cage while restricting food.t-test or two-way analysis of variance for comparisons between two groups. In all cases, two-tailed probability less than 0.05 (P < 0.05) was considered statistically significant. The details of each statistical analysis for each experiment are presented in the corresponding figure legends. The statistical analyses were conducted using Prism 8.0 software  or SPSS Statistics 26.0 software .All of the results are expressed as mean ± standard error of the mean. For statistical comparisons, we applied two-tailed unpaired Student’s n = 9) compared with conventional CUBIC  on day 10 (P < 0.05). With the aid of the optimized OSP-CUBIC protocol, high transparency was achieved in the adult mouse whole kidney, which is crucial for further kidney imaging and analysis.The kidney is a heme-rich organ, and obtaining high transparency is difficult. To develop an efficient tissue clearing protocol for whole-kidney imaging, we began to optimize classic CUBIC tissue clearing methods . We intrAfter OSP-CUBIC, the optically cleared and 3D-immunostained (sympathetic nerves stained with anti-TH antibody and vasculature labeled with FluoSphere) adult whole kidney was subjected to microscopic imaging . The chaX–Y plane and 3D-reconstructed images  at the endpoint of 8 weeks  compared with sham mice (n = 8) compared with the sham group  in MTAC mice compared with the sham group . The M-m 8 weeks . After 8ham mice . TerminaP > 0.05; n = 8) while compared to sham mice  were obtained by the production of total area on each section and sectioning thickness   . The ratIn the present study, we optimized OSP-CUBIC clearing, whole-mount kidney 3D immunostaining, and high-throughput observation by LSFM and confocal microscopy. We simultaneously visualized the high-resolution 3D structure of the whole-mount renal microvasculature, the glomerulus, and accompanying wrapped traveling sympathetic nerves of the mouse kidney. Further quantification by 3D analysis revealed that renal dysfunction occurred and sympathetic nerve hyperinnervated before structural and morphological changes after pressure overload-induced HF. This study established a novel 3D visualization protocol for kidney research.It is commonly difficult to acquire in-depth and comprehensive information about the kidney at the whole-mount scale. In a previous study of renal innervation under conditions of kidney disease, to acquire satisfactory images, the researchers performed immunostaining, imaging, and analyses using a half-cut kidney after kidney clearing . We moveThe kidney is abundantly innervated by sympathetic nerves, renal sympathetic nerve fibers release norepinephrine, adenosine triphosphate and neuropeptide Y in the kidney, and the effects of norepinephrine on adrenoceptors most robustly affecting renal function . So, symWith regard to CRS, acute and chronic HF is known to cause renal abnormality . VariousWe optimized a whole-mount kidney OSP-CUBIC clearing, 3D-immunostaining, image acquisition, and 3D analysis system and successfully visualized the 3D structure of the renal microvasculature, the glomerulus, and accompanying sympathetic nerves. We observed sympathetic hyperinnervation at early stages after pressure overload-induced HF in mice. This newly developed whole-organ tissue clearing and imaging method has great potential for further studies of kidney diseases and can provide comprehensive information at the whole-mount scale. This method can also be performed with other heme-rich organs, such as the heart, for which achieving satisfactory clearing and imaging of the whole organ is difficult.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by China-Japan Friendship Hospital.CW performed the primary experiments, acquired and analyzed the data, and wrote the manuscript. FY and ML performed some of the LSFM imaging and image processing. YT, ZG, YC, YW, and QL performed some of the animal experiments. CY, YF, and MW designed some of the experiments. JZ, XL, YG, and WK equally designed the study, analyzed the data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fibromyalgia is characterized by chronic widespread pain, and more than half of patients with fibromyalgia report that weather-related variables aggravate their symptoms. However, the differences in actual symptoms have not been measured between those with and without weather sensitivity. The present study aimed to investigate whether weather sensitivity associated with the minimal clinically important difference values of quality of life in patients with fibromyalgia, between those with and without weather sensitivity.Sixty-four consecutive outpatients with fibromyalgia on their first visit to our tertiary center were included. Weather sensitivity was measured using self-perceived symptoms. Pain intensity was measured using the 0–10 Numerical Rating Scale (NRS). Quality of life was measured using the Euro Quality of life-5 Dimensions-3 level (EQ-5D-3L) scale. The variables were subjected to univariable and multivariable analysis using the EQ-5D-3L scale.The mean age of the patients was 50 years. Forty-eight patients (75%) were women. The mean EQ-5D-3L score was 0.55. Thirty-seven patients (58%) reported weather sensitivity. In univariable analysis, the welfare recipient, weather sensitivity, and NRS values were associated with EQ-5D-3L scale scores. In multivariable analysis, NRS value and weather sensitivity were independently associated with EQ-5D-3L scale scores. The NRS and EQ-5D-3L scale scores were significantly worse in those with weather sensitivity than those without weather sensitivity. The difference in NRS values was less than 1.5 points between groups. The differences in EQ-5D-3L scale scores were 0.16 points between groups.Weather sensitivity was significantly associated with quality of life in patients with fibromyalgia. There was an association with weather sensitivity and the minimal clinically important difference values of quality of life in patients with fibromyalgia. The presence of weather sensitivity could have a key role in the quality of life in patients with fibromyalgia.The online version contains supplementary material available at 10.1186/s41927-021-00185-4. Fibromyalgia is a disease characterized by chronic widespread pain without a known cause or cure –4. The pPotential factors that aggravate fibromyalgia symptoms are weather-related variables, physical activity, emotional distress, and sleep disturbance , 15. SpeThe present study aimed to investigate whether weather sensitivity associated with the minimal clinically important difference values of quality of life in patients with fibromyalgia, between those with and without weather sensitivity.All methods were carried out in accordance with relevant guidelines and regulations. This study was approved by the Ethics Committee of Hayaishi Hospital, and all study participants provided written informed consent. The sample size was calculated using the G*Power software . Based on an effect size of 0.3, –25 the mData were retrospectively collected from medical records of consecutive outpatients with fibromyalgia during their first visit to our tertiary center. Inclusion criteria were: 1) age > 20 years; and 2) a diagnosis of fibromyalgia by a medical doctor, based on the American College of Rheumatology 2010 criteria . The diaDemographic data, including age, body mass index, sex, marital status, and welfare recipient were collected during the first visit to our tertiary center. Weather sensitivity, pain intensity, and quality of life were measured as described below.Weather sensitivity was routinely taken for all patients in our institution. Weather sensitivity was measured using self-perceived symptoms –19. SubjPain intensity was measured using the 0–10 Numerical Rating Scale (NRS) . The scaQuality of life was measured using the Euro Quality of life-5 Dimensions-3 level (EQ-5D-3L) scale, a generic scale used worldwide that assesses health in five dimensions: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression , 11. Eacp < 0.2 in the univariable analysis. Four variables were analyzed in the multivariable analysis for EQ-5D-3L scale score: marital status, welfare recipient, weather sensitivity, and the NRS values. Multicollinearity of the variables was also assessed (correlation coefficient < 0.9).All continuous data were expressed as means and standard deviations. The normality of the distribution was evaluated using the Shapiro-Wilk test for continuous variables. The correlation of EQ-5D-3L scale score with each variable was analyzed using the Pearson correlation coefficient test. The categorical variables included a dummy variable for the analysis. Multivariable analysis was used to investigate variables with P values < 0.05 were considered significant.All data were statistically analyzed using the SPSS 26.0 J program, and Patient characteristics are shown in Table Correlations between the EQ-5D-3L scale scores and independent variables are shown in Table The results of multivariable analysis for the EQ-5D-3L scale scores are shown in Table Table The present study demonstrated that weather sensitivity was significantly associated with quality of life in patients with fibromyalgia. The findings suggested that the presence of weather sensitivity could have a key role in the quality of life in these patients.Fibromyalgia induces pain and reduces the quality of life , 13. PreMany patients report that weather sensitivity aggravates pain, –19 consiInterventions in combination and organization would benefit fibromyalgia –4. TreatThere are several limitations to the present study. First, weather sensitivity was measured using only self-perceived symptoms using a binary scale, not a rating scale or actual local weather situations. Although the weather sensitivity using a binary scale is shown in one of the major aggravating factor in patients with fibromyalgia, , 16 therWeather sensitivity, followed by pain intensity, was significantly associated with quality of life. There was an association with weather sensitivity and the minimal clinically important difference values of quality of life in patients with fibromyalgia. This suggests that weather sensitivity could play a key role in the quality of life in patients with fibromyalgia. Ascertaining the presence of weather sensitivity has the potential to be useful in estimating the level of suffering experienced by patients with fibromyalgia.Additional file 1: Supplement Fig. 1. Difference of HADS-Depression subscale between those with and without weather sensitivity. Values are means of the HADS-Depression subscale score, and the error bar shows standard deviations (n = 51). The HADS-Depression subscale scores in those with weather sensitivity were significantly worse than those without weather sensitivity. * Significance level was set at < 5%. HADS, Hospital Anxiety and Depression Scale.


Mesenteric ischemia is a severe and potentially lethal event. Assessment of intestine perfusion is eminently depending on the skills, and the experience of the surgeon. Thus, the therapy is biased by the right evaluation. Aim of this study is to determine the applicability, and the usefulness of fluorescent-imaging (FI) with indocyanine green (ICG) in a porcine model of mesenteric ischemia. Second end-point is the verification of a visual and quantitative assessment tool of the intestinal perfusion.In 18 pigs  an occlusion of a side-branch of the mesenteric artery was performed for 3 , 6 , and 10 hours . After reperfusion a 60 minutes observation period was carried out. 3 regions of interest were defined: ischemic bowel (D1), transitional zone (D2), and non-ischemic bowel (D3). ICG-FI was performed during baseline (T0), occlusion (T1), reperfusion (T2) and after an observation period of 60 minutes (T4).All experiments could be finished successfully. ICG-FI was assessed using assessment of background-subtracted peak fluorescence intensity (BSFI), slope of fluorescence intensity (SFI), and a baseline adjusted ratio of both parameters. ICG-FI confirmed loss of perfusion in D1, decreased perfusion in D2, and increased perfusion in D3. After reperfusion ICG-FI increased in group 2 due to a severe tissue damage resulting in a capillary leakage. In group I ICG-FI was equal to baseline values indicating the totally reversible loss of perfusion.Using ICG-FI to estimate intestine perfusion after different durations of ischemia is viable using a porcine model of mesenteric ischemia. Even small differences in perfusion can be reliably determined by ICG-FI. Thus, ICG-FI is an encouraging method to evaluate intestine perfusion intraoperatively. Mesenteric ischemia is still a lethal disease. Fast diagnosis and therapy results in an improvement of prognosis. The high mortality of this disease, ranging between 60–80%, has not changed significantly in the last few years –4, despiDirect surgery is commonly recommended as the treatment of choice. Previous diagnostics often only delay proper treatment . During Still there is no standardized technique to evaluate tissue perfusion status during mesenteric ischemia. Indocyanine green fluorescent imaging (ICG-FI) is a technology for visual and quantitative evaluation of tissue perfusion in various fields –15. CurrThis study was carried out in stringent conformity with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was authorized by the Committee on the Ethics of Animal Experiments of the Authority for Health and Consumer Protection Hamburg . The experiments were performed under deep sedation. All attempts were made to minimize suffering.The experiments were performed in 18 pigs of either sex weighing 54.2 +/- 2.9 kg at the Institute for Surgical Research, University Center Hamburg-Eppendorf .2O with a positive end-expiratory pressure of 7 cm H2O at 16 breaths per minute using 30% oxygen. A central venous catheter was placed into the right jugular vein for volume substitution, and central venous pressure monitoring.Premedication and anesthesia was performed as previously described . BrieflyA 4F arterial catheter was inserted into the right femoral artery for continuous hemodynamic monitoring documented by the PiCCO device .Midline laparotomy was performed. A standardized model for mesenteric ischemia was previously established in an unpublished pilot study. Briefly, a huge branch of the superior mesenteric artery (SMA) was dissected in the meso-ileum next to the ileocecal junction. Due to huge venous and arterial collateralization, a complete dissection of the depending mesentery was performed, including the mesenteric vein and its venous weave . For stePigs were randomized into 3 groups with total ischemia duration of 3 , 6 , and 10 hours . 3 regions of interest (ROIs) were defined in ischemic (D1), transitional (D2), and control areas (D3) .Hemodynamic parameters, and metabolic parameters were documented hourly. Semi-objective assessment of each area was performed hourly by two experienced visceral surgeons using a macroscopic score including color, peristalsis, edema formation, and mesenteric hemorrhage. The degree of ischemia was categorized into none/mild (<6 points), moderate (6–9 points), and severe (>9 points).ICG-FI was performed with the FCI system (LLS GmbH) developed by the institution of laser technology of the university Ulm . ICG-FI The technique of ICG-FI and data analysis was previously described in detail by Detter et al 2007 . BrieflyTo calculate BSFI from the time-dependent fluorescence intensity curves, the initial intensity value before the injection of ICG was subtracted from the peak fluorescence intensity during the first passage of the dye through the bowel segment .This parameter is represented by the maximal slope during the increase of the time-dependent fluorescence intensity induced by the first wave of the dye, which passes the capillaries of the bowel tissue.Each ICG-FI sequence was recorded online for 120 seconds with real-time digitizing. BSFI and SFI data were analyzed offline directly after the images were recorded with a customized software package (LLS GmbH).Representative specimen of the bowel in the ischemic, transitional, and control area were removed, and thereafter stored in 3.5% buffered formalin. All specimens were routinely processed and embedded in paraffin. 5-μm slices were stained with hematoxylin and eosin. And examined blinded to the treatment using an established scoring system .2, fixed effects of group, areal, time and areal-by-time were tested, accounting for repeated measures over time within areal-by-animal. For the dependent variable “flow” and the hemodynamic dependent variables CO, CI, HR, MAP, SVR, GEDV and GVD a fixed effect for time was fitted and repeated measures over time within animal were accounted for. Non-significant fixed effect terms were excluded from the models using a hierarchical stepwise-backwards approach. For repeated measures, an autoregressive covariance structure was employed while a variance components covariance structure for estimating random intercepts. Time was treated as a categorical variable. A value of p<0.05 was considered statistically significant. Statistical analysis was performed with the SPSS statistical software package 23.0 .Data of continuous variables were presented as means and standard deviations (SD) across animals. Data distribution was visually assessed by histograms and box plots. The variables BSFI, SFI, SFI-ratio and BSFI-ratio were ln-transformed prior to further analyses to minimize skewness and heteroscedasticity. The means of these ln-transformed variables, along with their 95% confidence intervals, were graphically presented by group, location and time. Category counts of variable “macro score” were also graphically presented by group, location and time. Data were then subjected to mixed model analyses to take repeated measurements within animals into account. For the dependent variables BSFI and SFI, we fitted initial models containing fixed effects of group, areal, time, and areal-by-time. Random intercepts for raters and animals were fitted and repeated measures over time within areal-by-animal-by-rater were accounted for. For the dependent variables tpOExperiments have been performed successfully in all 18 pigs. None of the pigs had to be excluded.Hemodynamics were stable between the groups throughout the whole experiment without significant differences between the groups. However, there were slightly higher cardiac output values at baseline in group III, while systemic vascular resistance was highest in group I (n.s.).TTFM flow was comparable in all 3 groups during T0 (69.7± 16.5 ml/min), decreased to 0±0ml/min during complete occlusion, and increased immediately after reperfusion to highest values . Compensatory hyperemia was highest in group 3 and lowest in group 1. Values remained stable over 60 minute observational period.Macroscopic evaluation showed a worsening of bowel viability in all groups during ischemia. During occlusion, bowel viability in the ischemic area D1 was 11.4 ±1.8 after 3 h in group I, 11.67±1.03 after 6 h in group II, and 12.25±0.96 after 10 h in group III, while maintaining baseline values (4.0±0) in the non-ischemic area D3 in all groups (p<0.05). 60 minutes after reperfusion values in group I decreased to 7.0 ±1.41, in group II to 9.33±1.86, and remained at 12.50±1.25 in group III (p<0.05). Values of transitional zone D2 were significantly worse than in the non-ischemic area D3 (p<0.05) during occlusion. However, macroscopic Score in D2 was significantly better than the ischemic area D1 at the same time , as well as during 60 minutes after reperfusion . While macroscopic aspect of the bowel tissue improved again after reperfusion in the 3 h and 6 h group, irreversible severe damage was evaluated after 10 h of ischemia (p<0.05).Histopathologic examination was performed using the score described previously by Haglund  and ChiuVisual assessment of the ICG-FI revealed homogeneous perfusion of the corresponding bowel in all areas at baseline. Occlusion of the dissected SMA branch resulted in a total perfusion defect with no visible fluorescence signal in the corresponding bowel (D1). Visual assessment and evaluation of bowel viability after reperfusion was difficult in all areas, due to variable degree of residual fluorescent signals, depending on the period of ischemia.The impairment of perfusion was quantified by BSFI and SFI obtained with ICG-FI.Baseline (T0). Baseline values measured by SFI showed incongruent results in between the groups with significantly higher values in group III . The ROIs at baseline showed in all groups significantly higher ICG-FI in D3 (control), and lowest in D2  .Occlusion (T1). Under occlusion ICG-FI decreased significantly in D1 (ischemic) , and also in D2  , while D3 (control) showed similar, but slightly elevated ICG-FI values compared to baseline .Reperfusion (T2). After reperfusion D1 (ischemic) showed enhanced ICG-FI in all groups compared with occlusion , while SFI was lowest after 3 hours of ischemia (group I), and highest after 6 hours of ischemia (group II). ICG-FI in D2  was significantly impaired compared to baseline (p<0.05), but significantly improved compared to occlusion . ICG-FI in D3 remained high also after reperfusion  ( = n.s.) .Baseline (T0). BSFI at baseline showed similar results in group I and II (p = n.s.), while ICG-FI in group III was significantly higher .Occlusion (T1). Under occlusion, BSFI confirmed SFI data. ICG-FI in D1 (ischemic) and D2  decreased significantly (p<0.05), while ICG-FI in D3 was significantly elevated compared to D1 and D2, and similar to baseline .Reperfusion (T2). ICG-FI in D1 (ischemic) after reperfusion showed significantly enhancement compared to occlusion . Similar to SFI results, group III showed significantly higher ICG-FI compared to group I and group II after reperfusion (p<0.05). In contrast to SFI findings, group I showed similar ICG-FI levels like group III using BSFI analysis. BSFI results in D2  were comparable in all groups with highest levels in group III . Results were comparable to baseline values, and improved compared to occlusion. ICG-FI in D3 (control) remained high with highest values in group III   .To avoid the impact of differences in hemodynamics in between the groups, a ratio to baseline values for each group at occlusion and reperfusion was chosen. Thus, values of 1 are equal to baseline, values > 1 indicate higher ICG-FI, values <1 of lower ICG-FI.Occlusion (T1). Under occlusion SFI ratio reflected perfusion deficit in D1 in the different groups . D2 showed impaired perfusion in all groups , while D3 showed hyper intensity ).Reperfusion (T2). After reperfusion perfusion areal D 1 showed nearly equal intensity to baseline in group I , while group 2 showed more than doubled values . Interestingly Group 3 had similar results to group I with moderate hyper intensity . D2 and D3 showed hyper intensity after reperfusion in each group (ch group .Occlusion (T1). Perfusion deficit in D1 is well demonstrated , D2 showed reduced ICG-FI than baseline values , and D 3 revealed hyper intensity , thus, lower than SFI Ratio. After Reperfusion D 1 ICG-FI assimilated again to baseline value in group I , and group III , while group II showed hyper intensity . D2 and D3 revealed hyper intensity after reperfusion in each group (ch group .Acute mesenteric ischemia is still a life-threatening disease with a high mortality . EvaluatThis study focused on evaluation of ICG-FI in bowel assessment after different periods of ischemia, trying to predict tissue damage and comparing it to histopathological findings.ICG-FI to evaluate bowel ischemia has been published in small case series earlier –30. The Although ICG-FI is an increasingly popular method and is also widely used for determination of perfusion status in different procedures, so far there have only been a few attempts to carry out a quantification. In an experimental study inducing mesenteric ischemia of 60 minutes in a rat model Toens et al. compared ICG-FI to radioactive microspheres. They evaluated the increase of fluorescence by using the ROI technique. The results of the ischemic parts were compared to control regions. Although their results turned to be inhomogeneous compared to the microsphere technique, ICG-FI was concluded to be a feasible, reliable, and valid technique for mesenteric blood flow assessment . BehrendIn a comparable study by Matsui a 30 minutes ischemia was induced in a 2–12 cm bowel segment. Quantification was made by analyzing the absolute fluorescence intensity and the contrast-to-background-ratio. In conclusion, a clinical assessment had high sensitivity, but relatively low specificity. Thus, prediction of survival remained unsecure.Remarkable, quantitative assessment tools like the maximum fluorescence intensity resulted in only a slightly better bowel evaluation, compared to visual evaluation .In another study, Diana et al. compared ICG-FI to metabonomics, lactate levels, and histopathologic findings using a real time assessment in a porcine model. They used the time to peak in ischemic and non-ischemic areas of intestine for quantification after 15 minutes of ischemia. With their results, they clarify the effectiveness of the ICG-FI . In a suIn contrast to existing animal studies, the present study evaluates ICG-FI after significantly longer ischemic periods, which are closer to everyday routine. ICG-FI confirmed a significant difference of tissue damage comparing 6 and 10 hours of ischemia, in correlation to histological findings. The clinical interpretation of bowel quality after 10 hours is obvious. The evaluation of tissue damage after 6 hours of ischemia is still challenging. Therefore, quantification of ICG-FI using SFI and BSFI is a helpful tool to predict tissue damage, especially if the visual assessment is difficult. As we could show by comparing to microscopic findings, a hyper intensity in Group 2 dues to a cellular damage causing capillary leakage with successive ICG pooling. This ICG pooling may cause a misjudgment of the perfusion status of the bowel. Already damaged tissue can be estimated as optimal perfused by visual evaluation alone. The pooling stays longer due to loss of epithelial integrity. Excellent perfused tissue has a immediate influx of ICG, and a gradual, but obvious outflow.To reduce the effect of hemodynamic differences, a ratio to baseline values was established. All side effects, that influence hemodynamics, such as cardiac output, blood pressure, vascular resistance, volume load, catecholamine substitution also affect ICG-FI results and may lead to misinterpretation. These side effects and hemodynamics are important for accurate measurements. In clinical routine a stable experimental is not achievable. Thus, a ratio to a baseline value, or correlation to a non-ischemic area during the same measurement is mandatory for correct interpretation. Until now, the influence of hemodynamics is undervalued.SFI and BSFI are conclusive evaluation parameters using ICG-FI for detection of tissue viability. Further studies in humans are mandatory to determine the predictive value of ICG-FI assessment.This experimental study in a porcine model of mesenteric ischemia gives evidence that ICG-FI is a promising tool for bowel viability assessment after different periods of ischemia. Solely macroscopic evaluation is possible, but not sufficient. Additional quantitative assessment is useful to support intraoperative decision making. Nevertheless, hemodynamic status have to be taken into consideration to avoid misinterpretation of ICG-FI. Thus, observational studies in humans are intended to further consolidate the findings.


In addition, Ki-67 overexpression was significantly associated with a pathological T stage ≥ 2 (p = 0.027) and tumour necrosis (p = 0.016). In the multivariable analysis, a positive PD-L1 expression was significantly correlated with worse cancer-specific survival . However, there was no predictive value using a combination of PD-L1 expression and Ki-67 overexpression as a prognostic predictor. Compared with Ki-67 overexpression, a positive PD-L1 expression with CPS ≥ 10 was a stronger independent prognostic factor for CSS in patients with UTUC.We retrospectively enrolled 102 patients with upper tract urothelial carcinoma (UTUC) who underwent radical nephroureterectomy to examine the prognostic value of Ki-67 and programmed cell death ligand-1 (PD-L1). Then, we performed PD-L1 and Ki-67 immunohistochemical staining on whole tissue sections. The cut-off value of PD-L1 positivity was a combined positive score (CPS) ≥10 and the Ki-67 overexpression was 20%. Among the 102 patients, 16.7% and 48.0% showed positive PD-L1 expression and Ki-67 overexpression, respectively. A CPS ≥10 was significantly associated with a higher pathological T stage ( Upper tract urothelial carcinoma (UTUC) accounts for only around 5% of all urothelial carcinoma (UC) cases . HoweverBiomarkers may be crucial in predicting a UTUC prognosis. Programmed cell death-1 positivity was found to have an independent prognostic value of cancer-specific survival (CSS) outcomes in patients with UTUC receiving extirpative surgery . MoreoveBetween 2013 and 2018, a total of 102 patients with UTUC who underwent radical nephroureterectomy (RNU) through laparoscopic or traditional methods were retrospectively enrolled in this study. We excluded patients without PD-L1 or Ki-67 immunochemical stains, bladder cancer stage T2 or higher, and patients with distant metastasis at diagnosis or receiving neoadjuvant chemotherapy. Information regarding patients’ specimens was obtained from the Surgical Pathology Database of Kaohsiung Chang Gung Memorial Hospital (KCGMH). This study was approved by the Institutional Review Board of KCGMH .Lymph node staging was performed by radiologists or the treating physician based on the computed tomography (CT) scan findings. Lymph node dissection (LND) was performed during the operation when enlarged lymph nodes were noted. The pathological tumour stage was determined according to the eighth edition of the American Joint Committee on Cancer’s staging system for cancer of the renal pelvis and ureter.Locoregional recurrence was defined as the presence of a recurrent lesion at the previous operation site or the evidence of retroperitoneal lymph node enlargement on the CT scan during follow-up. Distant metastasis was defined as the evidence of suspicious lesions on radiological images. In addition, recurrent bladder cancer was determined on the basis of pathological reports. The history of bladder cancer and the cause of death were determined through a chart review.Specimen staining was performed by a specialised pathologist (SMZ). The tissue samples submitted for the PD-L1 study were fixed in formalin, embedded in paraffin, and sectioned into 3-μm thickness. The immunohistochemical examination of PD-L1 was performed using a Dako anti-PD-L1 (clone 22C3) mouse monoclonal primary antibody purchased from Agilent Technologies . The detection system used was the EnVision FLEX Visualisation System . Immunostaining was performed using a Dako Autostainer Link 48 . The specimens for Ki-67 immunostaining were fixed in 10% formalin, embedded in paraffin, and sectioned into 1-μm thickness. The tissue sections were deparaffinised using xylene and a graded ethanol series and then heated in boiling 0.01-M citrate buffer (pH 6.0) for 15 min in a microwave oven for antigen retrieval. Subsequently, the sections were incubated with the primary mouse monoclonal anti-Ki-67 antibody  diluted in the antibody diluent reagent solution  at room temperature for 60 min, rinsed with a buffer, amplified using the Quanto Amplifier and Quanto HRP polymer , labelled with DAB chromogen , and then counterstained with haematoxylin. A negative reagent control was set, in which the patient tissue was processed using the same steps, except for incubation with the primary antibody. A small section of the tonsil tissue attached on each experimental slide was used as the positive  and negative (mature squamous epithelium) tissue control.The combined positive score (CPS) was used to examine the PD-L1 expression in the tissue sections. The CPS was defined as the ratio of the number of PD-L1-stained cells  to the total number of tumour cells. We performed a time-dependent receiver operating characteristic (ROC) curve and survival ROC curve by the SAS system to find the cut-off value of PD-L1 in this study. A CPS ≥ 10 was found to be the optimal cut-off value of this study CPS of <10 and ≥10 indicated negative and positive PD-L1 expressions, respectively .We visually calculated the Ki-67 index by counting the total number of stained tumour cells and the total number of tumour cells in each high-power field for a total of five high-power fields. The Ki-67 index was calculated as the number of stained tumour cells divided by the total number of tumour cells and then multiplied by 100. The cut-off value of Ki-67 overexpression was also calculated by the SAS system by the same methods. Ki-67 overexpression was considered when the samples exhibited a nuclear reactivity of ≥20% .p < 0.05. All statistical analyses were performed using SPSS .The relationships among the PD-L1 expression, Ki-67 overexpression, clinical characteristics, and pathological features were examined using the chi-square test or Fisher’s exact test. The Kaplan–Meier method was used to evaluate the survival function, and the differences were examined using the log-rank test. Univariable and multivariable Cox proportional hazard regression models by forward stepwise selection for the OS, as well as CSS, were used to assess the prognostic indicators—namely, sex, age at diagnosis, PD-L1 expression, Ki-67 overexpression, tumour node metastasis classification, and other clinicopathological characteristics. The level of significance was set at p = 0.006) and those with a Ki-67 index of ≥20 .Of the 102 patients, 18 and 49 exhibited positive PD-L1 expression and Ki-67 overexpression, respectively. The results are presented in p = 0.049), higher proportion of patients receiving palliative immunotherapy , and lower proportion of patients exhibiting bladder tumour recurrence after an operation during follow-up . A higher proportion of patients with a pathological T stage ≥ 2 showed Ki-67 overexpression . Furthermore, the patients with Ki-67 overexpression showed a higher proportion of tumour necrosis in specimens .A PD-L1-positive expression was associated with a higher T stage  or CSS  and a higher T stage were also found to be independent risk factors for a shorter OS. In addition, a positive PD-L1 expression did not show a significant correlation with the OS, and the combination of CPS ≥ 10 and Ki-67 ≥ 20% did not show significance. Furthermore, a positive PD-L1 expression , a lymph node ≥10 mm in size on a CT scan , receiving palliative chemotherapy , and a higher T stage were independent risk factors for shorter CSS. The combination of positive PD-L1 and overexpression of Ki-67 was not correlated with CSS after the analysis.In the multivariable analysis using forward stepwise selection for the OS , age oveAccording to the 2020 European Association of Urology (EAU) guidelines, several factors are associated with a poor prognosis of UTUC, including a high pathological stage, older age, multifocality, and hydronephrosis . These gA prospective study was conducted by Krabbe, L.M. et al. to investigate the prognostic value of Ki-67 in patients with UTUC who underwent RNU . This stIn our study, we found that Ki-67 overexpression was not associated with CSS and OS in the multivariable analysis. Although we enrolled Asian patients in our study, our results differed from those reported for Japanese and Korean patients. A cohort study examined and compared the pathological characteristics and behaviours of patients with UTUC between Taiwan and Japan [p = 0.030,). Therefore, these results also implied that PD-L1 positivity may have a stronger association with a worse prognosis than Ki-67 overexpression.In this study, we investigated the Ki-67 overexpression and PD-L1 expression simultaneously. A shorter follow-up duration was found both in patients with PD-L1-positive expression and Ki-67 overexpression, which may be attributed to the poorer prognosis in these patients. Additionally, a CPS ≥ 10 was associated with a higher pathological T stage. Ki-67 overexpression was only associated with pathological T2 or higher. However, the linear-by-linear association test showed that Ki-67 overexpression was associated with a higher T stage . The prevalence of upper tract urothelial carcinoma is relatively low in comparison with bladder cancer. To minimise the specimen heterogeneity in this study, we only enrolled patients meeting the selection criteria, and this may have further minimised our sample size. Second, the lymph node status of most patients was determined on the basis of images. In addition, we did not routinely perform LND during RNU, because the benefits of LND are still under debate. We performed LND only if suspicious enlarged lymph nodes (>10 mm) were observed on the CT images before RNU or palpable enlarged lymph nodes were observed during the operation. Therefore, some nodal metastasis bias might have occurred. Third, given the relatively small sample size and few events, we failed to include all the established prognostic factors of OS or CSS in the multivariable analysis. However, we combined the prognostic variables with the most impact, such as immunochemical staining and the T and N stages, to evaluate the OS and CSS. Due to its higher prognostic value for CSS, PD-L1 may serve as a potential marker in treatment-related decision-making. The predictive value of PD-L1 should be examined in further prospective large-scale studies.The results indicated that a positive PD-L1 expression with CPS ≥ 10 was significantly associated with the pathological T stage. Additionally, a Ki-67 overexpression of ≥20% demonstrated a significant association with pathological T ≥ 2 and tumour necrosis. Compared with Ki-67 overexpression, positive PD-L1 expression was found to be a stronger independent prognostic factor for CSS in patients with UTUC.